Susceptibility of Human Lymphoid Tissue Cultured ex vivo to Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Infection

Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus

Susceptibility of Human Lymphoid Tissue Cultured ex vivo to Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Infection

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored RESULTS: XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. CONCLUSIONS: Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus

Endorectal Magnetic Resonance Imaging and Spectroscopy Are Useful for Selecting Candidates for Biopsy among Patients with Persistently Elevated Prostate Specific Antigen

To evaluate the efficacy of endorectal Magnetic Resonance Imaging (MRI) and Magnetic Resonance Spetroscopic Imaging (MRSI) combined with total prostate-specific antigen (tPSA) and free prostate-specific antigen (fPSA) in selecting candidates for biopsy. Subjects and Methods: 246 patients with elevated tPSA (median: 7.81 ng/ml) underwent endorectal MRI and MRSI before Transrectal Ultrasound (TRUS) biopsy (10 peripheral + 2 central cores); patients with positive biopsies were treated with radical intention; those with negative biopsies were followed up and underwent MRSI before each additional biopsy if tPSA rose persistently. Mean follow-up: 27.6 months. We compared MRI, MRSI, tPSA, and fPSA with histopathology by sextant and determined the association between the Gleason score and MRI and MRSI. We determined the most accurate combination to detect prostate cancer (PCa) using receiver operating curves; we estimated the odds ratios (OR) and calculated sensitivity, specificity, and positive and negative predictive values. Results: No difference in tPSA was found between patients with and without PCa (p = 0.551). In the peripheral zone, the risk of PCa increased with MRSI grade; patients with high-grade MRSI had the greatest risk of PCa over time (OR = 328.6); the model including MRI, MRSI, tPSA, and fPSA was more accurate (Area under Curve: AUC = 95.7%) than MRI alone (AUC = 85.1%) or fPSA alone (AUC = 78.1%), but not than MRSI alone (94.5%). In the transitional zone, the model was less accurate (AUC = 84.4%). The association (p = 0.005) between MRSI and Gleason score was significant in both zones. Conclusions: MRSI is useful in patients with elevated tPSA. High-grade MRSI lesions call for repeated biopsies. Men with negative MRSI may forgo further biopsies because a significantly high Gleason lesion is very unlikel

GFP-expression in DERSE XMRV indicator cells infected with tonsil supernatants.

<p>DERSE XMRV indicator cells were infected with tonsil supernatants harvested at day 16 and 22 post-infection (donor 402, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037415#pone-0037415-g003" target="_blank">Figure 3B</a>) and with a XMRV stock (22Rv1 supernatant) as positive control. A) DERSE-GFP positive cells were visualized by conventional phase contrast microscopy and fluorescence microscopy. Phase contrast image (left panel), GFP fluorescence image (central panel) and GFP/phase contrast merge image (right panel). Data are representative of two independent experiments. B) Quantitative assessment of DERSE GFP-positive cells. After 7 and 10 days post-infection (D7 p.i. and D10 p.i.) DERSE infected cells were harvested and the percentage of GFP-expressing cells was analyzed by FACS analysis.</p

Hypermutation of XMRV in infected human lymphoid tissue.

<p>DNA from tonsillar tissue infected with XMRV was amplified, cloned and sequenced. A) Graphic representation of the changes (compared to XMRV VP42) presents in the XMRV <i>gag</i> region. The mutations presents in clones from 5 different donors are shown. Each mutation is denoted by a vertical line marked onto a single horizontal line representing the entire amplicon, color coded with respect to dinucleotide context: GG→AG (red). GA→AA (cyan), GC→AC (green), GT→AT (magenta), and non G→A (black). Analysis was performed using the HYPERMUT program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037415#pone.0037415-Rose1" target="_blank">[91]</a>. B) Preferences of nucleotide substitutions from all the obtained sequences, “n” denotes the total number of sequenced base pairs. C) Summary of the observed changes at the different dinucleotide context. “n” denotes the number of mutations;^aPercentage of the total number of changes; ^bPercentage of the number of G-to-A changes.</p

Re-infection of human lymphoid tissue with tonsil supernatants.

<p>New histocultures from two different donors were prepared and infected at day 0 and 1 with a XMRV stock (22Rv1 supernatant) and supernatants harvested at day 16 and 22 post-infection (donor 402, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037415#pone-0037415-g003" target="_blank">Figure 3B</a>). The positive control (22Rv1 supernatant) was cultured in the presence or absence of AZT. A) Proviral XMRV DNA analyzed by quantitative real-time PCR in tissue cells isolated after 28 days post-reinfection. Each sample was run in duplicate. B) Absolute proviral XMRV DNA content in cells migrating out of the tissue collected from days 7 to 28 post-reinfection. C) Kinetics of XMRV RNA released by lymphoid tissue into the medium analyzed by real-time qRT-PCR.</p

Quantitative time course of XMRV <i>gag</i> sequences of <i>ex vivo</i> infected tonsillar tissue.

<p>Isolated DNA was amplified by quantitative real-time PCR and the absolute XMRV copy numbers were obtained by normalizing the results with the values obtained with the single copy CCR5 gene in each sample. Each sample was run in duplicate. A) Absolute XMRV DNA content in tissue cells, after 22 days of infection, in the presence or absence of two antiretroviral drugs, AZT and RAL. B) XMRV DNA content in cells migrating out of the tissue collected from days 2 to 22 post-infection in the presence or absence of the indicated inhibitors.</p